The purpose of these studies is to establish a better understanding of the energy metabolism of biological tissues, in vivo. Towards this goal, the laboratory concentrates on the use of minimally-invasive techniques to evaluate cellular energy metabolism of heart and skeletal muscle. The following major findings were made over the last year: 1) A mathematical network model has revealed a flaw in the consensus model of oxidative phosphorylation dealing with the kinetics of NADH oxidation. This result has focused our efforts in identifying the active binding sites for NADH and NADH metabolism in the matrix. In addition, we have shown that a very high affinity (< 10 uM) high gain control of respiration by ADP is possible if the mitochondria membrane potential is maintained during a work transition. This later result suggests a mechanism where by very small changes in cytosolic ADP could result in large changes in mitochondrial ATP synthesis as we have observed, in vivo, using 31P NMR techniques. 2) The binding sites that enhance NADH fluorescence in the mitochondria matrix are under investigation to understand NADH oxidation processes as well as validate NADH fluorescence imaging methodologies. We have isolated Site 1 from porcine heart mitochondria and established that it rapidly oxidizes NADH and forms oxygen free radicals. We were unable to identify a fluorescence frequency shift or lifetime change associated with NADH binding and oxidation by Site 1. These data suggest that Site 1 is not the matrix NADH binding site affecting NADH fluorescent properties. We are currently exploring the possibility that other dehydrogenases associated with Site 1 are responsible for the fluorescence enhancement in the intact mitochondrion. 3) Using the optical photolysis of NADH to photo-label associated matrix proteins, we have identified isocitrate dehydrogenase as the potential dominate matrix binding site. We are currently investigating whether matrix Site 1 and isocitrate dehydrogenase are associated to explain the strong coupling of NADH oxidation with fluorescence enhancement. 4) A screen of the mitochondria proteome and phosphoproteome is being undertaken using 2D gel electrophoresis, mass spectroscopy and radioisotope techniques. We have confirmed an extensive and dynamic phosphorylation of mitochondrial matrix proteins using 32-P in intact mitochondria. The phosphorylation of more than 20 proteins was observed in less than 10 minutes and was rapidly reversed by destroying the mitochondrial membrane potential. This is the first study that demonstrates a dynamic and widespread protein phosphorylation is occurring in the mitochondria matrix. Previously, the degree of protein phosphorylation in the mitochondria matrix was believed to be very limited to only a handful of proteins. This is a new signaling pathway that may provide insight into acute mitochondrial regulation in ATP synthesis as well as apoptosis control. 5) Minimally invasive, two photon excitation fluorescence microscopy (TPEFM) in intact animals is being used to study sub-cellular metabolic processes within cells under normal in vivo conditions using mitochondrial NADH and various exogenous fluorescent probes. We have further refined the multiphoton approach by the development of a periscope system to increase the degrees of freedom in applying the microscope objective on a living animal or human subject. We have also developed an optical gel to improve the coupling of the microscope objective to the animal. Currently the major issues are the correction of tissue motion on the micron scale to permit following of individual cellular regions during physiological perturbations. We are investigating different ventilation schemes as well as prospective and retrospective gating schemes to correct for physiological motion. 6) Using this multiphoton approach, the exterior surface of the porcine heart was investigated. The visceral epicardium was investigated and found to be an extensive network of elastin and collagen that spanned many millimeters over the surface of the heart. This layer interfered with optical measurements of the myocardial cells by extensive scattering and fluorescence interference from the elastin elements. We demonstrated, from various mechanical measures in the presence and absence of the visceral epicardium, that this 50 micron layer is a major passive mechanical element of the heart and may play an important role in several disease states of the heart.